Surveillance of Human Rotavirus in Wuhan, China (2011–2019): Predominance of G9P[8] and Emergence of G12

Rotaviruses are a major etiologic agent of gastroenteritis in infants and young children worldwide. To learn the shift of genotypes and genetic characteristics of Rotavirus A (RVA) causing diarrhea in children and adults, a hospital-based surveillance of rotavirus was conducted in Wuhan, China from June 2011 through May 2019, and representative virus strains were phylogenetically analyzed. Among a total of 6733 stool specimens collected from both children and adults with acute gastroenteritis, RVA was detected in 25.5% (1125/4409) and 12.3% (285/2324) of specimens, respectively. G9P[8] was the most common genotype (74.5%), followed by G1P[8] (8.7%), G2P[4] (8.4%), and G3P[8] (7.3%), with G9P[8] increasing rapidly during the study period. The predominant genotype shifted from G1P[8] to G9P[8] in 2012–2013 epidemic season. G12P[6] strain RVA/Human-wt/CHN/Z2761/2019/G12P[6] was detected in April 2019 and assigned to G12-P[6]-I1-R1-C1-M1-A1-N1-T2-E1-H1 genotypes. Phylogenetic analysis revealed that VP7, VP4, VP6, VP3, NSP1, NSP2, and NSP5 genes of Z2761 clustered closely with those of Korean G12P[6] strain CAU_214, showing high nucleotide identities (98.0–98.8%). The NSP3 gene of Z2761 was closely related to those of G2P[4] and G12P[6] rotaviruses in Asia. All the eleven gene segments of Z2761 kept distance from those of cocirculating G9P[8], G1P[8], and G3P[8] strains detected in Wuhan during this study period. This is the first identification of G12 rotavirus in China. It is deduced that Z2761 is a reassortant having DS-1-like NSP3 gene in the background of G12P[6] rotavirus genetically close to CAU_214.


Introduction
Group A rotavirus (RVA) is one of the leading etiological agents of diarrheal disease among children, and more than 120,000 deaths among children younger than five years were due to rotavirus in 2016, the fifth most fatal pathogen globally [1]. Vaccination is an essential strategy for the prevention and control severe diseases caused by rotavirus infection. Two rotavirus vaccines (Rotarix TM and RotaTeq ® ) are recommended for routine immunization of all infants by the World Health Organization (WHO) and have been introduced in more than 100 countries except for China [2][3][4]. A third rotavirus vaccine (Lanzhou Lamb rotavirus vaccine (LLR)) derived from a lamb G10P [12] rotavirus strain was licensed in 2000 and available in mainland of China [5]. Children in China were inoculated about 60 million doses of LLR during 2008-2014 [6].

The Case of G12 RVA Infection
The patient infected with G12P [6] RVA was a 59-year-old women who retired and was living in Jianghan District of Wuhan city. On early onset of gastroenteritis, the patient had diarrhea four times a day without vomiting or fevers. Then she visited the outpatient department of Wuhan Commercial Staff Hospital on 3 July 2019. After treatment with oral rehydration salts within three days, she recovered. The patient had not been vaccinated of LLR and none of the family members developed symptoms of gastroenteritis. She did not have any pets. Before the occurrence of diarrhea, she had neither gone out for tourism nor contacted with wild animals.

Strains
Collection

Structural Protein Genes
Phylogenetically, the VP7 gene of Z2761 was grouped into G12 RVA strains and clustered with those of strains detected in neighboring countries including Korea (CAU_214), Thailand (CHMN49-12) and Nepal (06N0440) with the minimum nucleotide identity of 98.4% and other human G12 strains reported worldwide such as in Europe, Africa, and the USA, showing nucleotide identity over 96.5% ( Figure 3). Pathogens 2020, 9, x 6 of 19

Structural Protein Genes
Phylogenetically, the VP7 gene of Z2761 was grouped into G12 RVA strains and clustered with those of strains detected in neighboring countries including Korea (CAU_214), Thailand (CHMN49-12) and Nepal (06N0440) with the minimum nucleotide identity of 98.4% and other human G12 strains reported worldwide such as in Europe, Africa, and the USA, showing nucleotide identity over 96.5% (Figure 3).   The VP6 genes of all the seven strains were assigned to the I1 genotype. Z2761 VP6 gene clustered closely with those of the strains from Korea (CAU_214), South Africa (MRC-DPRU1191), and European countries including Italy (ME659), Belgium (B4633), and Spain (SS96099331) with the minimum nucleotide identity of 98.7% ( Figure 5, I1c). The VP6 genes of E5365 and E6356 were close to those of Chinese strains (E2432, E3239, and JS2015) with minimum nucleotide identity of 99.0% ( Figure 5, I1b). The VP6 genes of E5867, E6398, L2448, and Z2768 were close to those of Chinese strains (R588 and Y128) and South African strains (3176WC and MRC-DPRU2130-05) with minimum nucleotide identity of 99.0% ( Figure 5, I1a). The VP6 genes of all the seven strains were assigned to the I1 genotype. Z2761 VP6 gene clustered closely with those of the strains from Korea (CAU_214), South Africa (MRC-DPRU1191), and European countries including Italy (ME659), Belgium (B4633), and Spain (SS96099331) with the minimum nucleotide identity of 98.7% ( Figure 5, I1c). The VP6 genes of E5365 and E6356 were close to those of Chinese strains (E2432, E3239, and JS2015) with minimum nucleotide identity of 99.0% ( Figure 5, I1b). The VP6 genes of E5867, E6398, L2448, and Z2768 were close to those of Chinese strains (R588 and Y128) and South African strains (3176WC and MRC-DPRU2130-05) with minimum nucleotide identity of 99.0% ( Figure 5, I1a). The VP1 and VP2 genes of all the seven strains were assigned to the R1 and C1 genotypes, respectively ( Figure S2 and Figure S3). The VP1 and VP2 genes of Z2761 clustered closely with those of European strains (RG177 and ss96099331) and African strains (KDH651 and 0050) with high nucleotide identity of 97.5-98.7% ( Figure S2, R1b and Figure S3, C1a). The VP1 genes of E5365, E5867, E6356, E6398, L2448, and Z2768 were close to those of strains from China and Thailand (JS2015, E3239, CU331-NR, and CU460-KK), with high nucleotide identities of 98.7-99.8% ( Figure S2, R1a). Similarly, the VP2 genes of the six strains mentioned above were close to those of Chinese and South African strains (Y128, R588, km15099, JS2015, E2432, and MRC-DPRU2130-05), with minimum nucleotide identity of 98.3% ( Figure S3, C1b).

Discussion
Comparing to the previous surveillance on rotavirus in Wuhan, the detection rate of RVA increased in adults, while being stable in children (χ 2 = 13.2, p < 0.01), and the rate was higher in children rather than in adults repeatedly [43][44][45]. When the data from other countries were compared, lower incidence (25.5%) of RVA in children was observed in Wuhan except for that in 13-24 month age group (40.0%), similarly in proportion to that reported in Southeast Asia (40.78%) [46,47]. In present study, major of specimens were collected from outpatients and most cases with relatively mild symptoms, which could be responsible for the lower incidence of RVA in children. Considering the highest incidence was in the 6-12-month age group, early introduction of routine rotavirus vaccination to children is desirable.
The incidence of rotavirus diarrhea in adults has been reported ranging from 2 to 22% of gastroenteritis cases in numerous countries [48,49]. In the present study, the incidence (12.3%) of

Discussion
Comparing to the previous surveillance on rotavirus in Wuhan, the detection rate of RVA increased in adults, while being stable in children (χ 2 = 13.2, p < 0.01), and the rate was higher in children rather than in adults repeatedly [43][44][45]. When the data from other countries were compared, lower incidence (25.5%) of RVA in children was observed in Wuhan except for that in 13-24 month age group (40.0%), similarly in proportion to that reported in Southeast Asia (40.78%) [46,47]. In present study, major of specimens were collected from outpatients and most cases with relatively mild symptoms, which could be responsible for the lower incidence of RVA in children. Considering the highest incidence was in the 6-12-month age group, early introduction of routine rotavirus vaccination to children is desirable.
The incidence of rotavirus diarrhea in adults has been reported ranging from 2 to 22% of gastroenteritis cases in numerous countries [48,49]. In the present study, the incidence (12.3%) of RVA was higher than that in Thai adults (8.7%) [50]. It is suggested that rotavirus gastroenteritis in adult population should be also prevented by any intervention in China.
Rotavirus vaccination is not included in China's national immunization program. The population of children under five years of age is about 300,000 in Wuhan. Of these, 3,000-25,000 infants received the vaccination annually since the year of 2005 when LLR had been introduced. Low coverage rates of vaccine may be responsible for the ongoing epidemics in Wuhan. The detection rate in children did not decrease after vaccination [43][44][45]. It was deduced that the influence of LLR on the prevalence and genotype of RVA in Wuhan was limited.
Phylogenetically, the VP7, VP4, VP6, VP3, NSP1, NSP2, and NSP5 genes of the G12P[6] strain Z2761 were close to those of the Korean G12P [6] strain (CAU_214), while all the structural and nonstructural protein genes of Z2761 kept distance from those cocirculating strains in Wuhan in this study period. Because of the limited epidemical information, the origin of infection and the route of transmission of Z2761 was not clear. Considering the phylogenetic characters of the whole genome of Z2761 and case information of the patient, this emerging G12P [6] RVA in China was hypothesized to be brought from rotavirus sharing a common ancestor with strain CAU_214. During transmission of G12P [6] RVA, NSP3 gene of DS-1-like human rotavirus was suggested to be reassorted via coinfection, resulting in the emergence of strain Z2761. To our knowledge, Z2761 is the first G12 strain detected in China. More information should be accumulated through intensive nationwide surveillance to monitor prevalence of G12 in China.

Specimens
A hospital-based surveillance of sporadic rotavirus diarrhea was conducted in Wuhan, China. Fecal specimens were collected from inpatients and outpatients in four hospitals (Renming Hospital of Wuhan University, Wuhan Commercial Staff Hospital, Wuhan the Sixth Hospital, and Wuhan Children's Hospital) from June 2011 through May 2019. All the specimens were stored at −80 • C. Fecal specimens were analyzed as part of routine surveillance of infectious diseases that had been approved by the ethics committee of Wuhan Centers for Disease Control and Prevention. Oral informed consent was obtained from the patients or guardians for all samples collected.

Investigation on The Case Infected G12 Rotavirus
Epidemiological information including the demographics, symptoms, and contact history was collected in the hospital. More information of the case infected G12 RVA was obtained by a telephone survey.

Detection of Rotavirus
Viral dsRNA was extracted from 400 microliters of 10% stool suspension with sodium dodecyl sulfate (SDS) and phenol and precipitated with ethanol [68]. RNA segments of rotavirus were separated by polyacrylamide gel electrophoresis (PAGE) and stained with silver nitrate [69].

Genotyping of RVA
Viral dsRNA was extracted from 200 microliters of 10% stool suspension by using the automatic nucleic acid extraction system NP968S with Nucleic Acid Extraction Kit (Jiangsu Tianlong Science and Technology Co. Ltd., Jiangsu, China) according to the manufacturer's instructions. Rotavirus G-type and P-type had been determined by nested reverse transcription-polymerase chain reaction (RT-PCR) from 2011 through 2015 [70][71][72][73].
The genotypes of VP7 and VP4 genes have been determined by analyzing the sequences of the first round PCR products since 2016 [70][71][72][73]. The nucleotide sequences of primers were listed in Table S3. PCR products were subjected to direct sequencing by Sanger method at Sangon Biotech (Shanghai) Co. Ltd. (Wuhan). The genotype of RVA was preliminarily assigned by the Basic Local Alignment Search Tool (BLAST).

Whole Genome Sequencing
The complete 11 segmented genes of whole genome were amplified by RT-PCR. The nucleotide sequences of primers were listed in Table S3 and included those reported in our previous study [63]. PCR products were subjected to direct sequencing by Sanger method.

Phylogenetic Analysis
Nucleotide sequence of each segment was assembled and edited by DNAMAN software. Genotype was determined by RotaC version 2.0, an online automated genotyping tool for RVAs [74]. The sequence identities were analyzed by Lasergene bio-information software (DNASTAR, Inc, Madison, Wisconsin, USA). Multiple alignments of the sequences were performed using MAFFT v7.471 (Kazutaka Katoh, Osaka, Japan) [75]. Phylogenetic analysis was conducted together with reference strain sequences obtained in the National Center for Biotechnology Information (NCBI) database by MEGA program version 7.1 [76]. The evolutionary history was inferred by using the Maximum Likelihood method, with 1000 bootstrap replicates, based on the best nucleotide substitution model with the lowest Bayesian information criterion (BIC) score in MEGA v7.1 [77]. Initial tree for the heuristic search was obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree was drawn to scale, with branch lengths represented by the number of substitutions per site.

Statistical Analysis
Statistical analysis was performed by SPSS version 18.0 (SPSS Inc., Chicago, IL, USA) software. Testing for the statistical significance was performed using the chi-square test. p-value less than 0.05 is considered significant.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-0817/9/10/810/s1, Table S1: Frequency of G-and P-genotypes of RVA detected in children and adults in Wuhan between 2011 and 2019. Table S2: Frequency of G-and P-genotypes of RVA detected in children in Wuhan between 2011 and 2019. Table S3: Primers used for RT-PCR and sequencing in this study. Figure S1: RNA patterns of G12P [6] rotavirus strain (Z2761) and the other six G9P [8] strains collected in March 2019. Lane 1, E6390; Lane 2, E6392; Lane 3, E6394; Lane 4, E6397; Lane 5, E6398; Lane 6, Z2757; Lane 7, Z2761. Figure S2: Phylogenetic dendrogram based on complete coding regions of the VP1 genes of representative RVAs. The best nucleotide substitution model is TN93 + G + I. Figure S3: Phylogenetic dendrogram based on complete coding regions of the VP2 genes of representative RVAs. The best nucleotide substitution model is TN93 + G + I. Figure S4: Phylogenetic dendrogram based on complete coding regions of the VP3 genes of representative RVAs. The best nucleotide substitution model is GTR + G + I. Figure S5: Phylogenetic dendrogram based on complete coding regions of the NSP1 genes of representative RVAs. The best nucleotide substitution model is GTR + G + I. Figure S6: Phylogenetic dendrogram based on complete coding regions of the NSP2 genes of representative RVAs. The best nucleotide substitution model is T92 + I. Figure S7: Phylogenetic dendrogram based on complete coding regions of the NSP5 genes of representative RVAs. The best nucleotide substitution model is T92 + G.